SNAP output for PDB entry 8eve [SNAP web server]

PDB-id

8eve; SNAP-derived features in text and JSON formats; DNAproDB

Class

replication,transferase-DNA

Method

X-ray (2.35 Å)

Summary

Human DNA polymerase eta insertion complex

Reference

Richie-Jannetta R, Pallan P, Kingsley PJ, Kamdar N, Egli M, Marnett LJ (2023): "The peroxidation-derived DNA adduct, 6-oxo-M 1 dG, is a strong block to replication by human DNA polymerase eta." J.Biol.Chem., 299, 105067. doi: 10.1016/j.jbc.2023.105067.

Abstract

The DNA adduct 6-oxo-M₁dG, (3-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct M₁dG. However, the effect of 6-oxo-M₁dG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-M₁dG. Using steady-state kinetics and analysis of DNA extension products by LC-MS/MS, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer-templates across from the 6-oxo-M₁dG adduct, with dGMP being slightly preferred. We also show primer-templates with a 3'-terminal dGMP or dAMP across from 6-oxo-M₁dG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-M₁dG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-M₁dG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site, but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-M₁dG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-M₁dG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability.